A disproportionately large number of mutations resulting in neurodevelopmental and neuropsychiatric disorders target synaptic proteins. Synapse remodeling and loss precede cell death in neurodegenerative disorders, and addictive drugs can alter circuit connectivity. The convergence of so many brain disorders at the synapse indicates that proper synapse structure and efficacy are critical to normal brain function. While multiple proteins have been implicated in synapse formation and elimination, the sequence of events leading to assembly/disassembly of the elaborate protein complexes that reside on both sides of the synapse has not been delineated. Resolving the steps leading to synapse formation in vivo has been limited by the difficulty of discretely labeling and simultaneously tracking the recruitment and assembly of individual synaptic components, which requires the ability to resolve multiple protein labels in discriminable colors while imaging over physiologically relevant timescales. Technology for robust, real-time monitoring of synapse assembly/disassembly in vivo would have enormous impact not only on our understanding of this fundamental feature of brain development and plasticity, but also in its application to the many disease models that derive from synaptic deficits. To tackle this imaging challenge, our goal is to develop high-resolution, high-throughput Temporal Focusing (TF) two-photon microscopy for large-volume imaging of synapse assembly/disassembly in vivo in the mouse brain. With this novel parallelized approach, we hope to achieve high-resolution imaging in vivo with 1-2 orders of magnitude higher throughput than point scanning, but with comparable resolution and signal-to-noise ratio (SNR). This would enable repeated imaging of entire dendritic arbors and their resident synapses over the short intervals necessary to observe synapse formation and elimination in real time, without the burden of anesthesia becoming intolerable. To pilot and validate the method, we will image the structural dynamics of excitatory and inhibitory post-synaptic scaffolding proteins distributed across the full dendritic arbors of Layer 2/3 (L2/3) pyramidal neurons in the developing mouse visual cortex. Further, we propose to combine our in vivo TF system with Magnified Analysis of the Proteome (MAP), a form of expansion microscopy, to interrogate the protein content of synapses for which we have a dynamic history from in vivo imaging. With the power of these combined approaches, our data can reveal, for the first time and in unprecedented detail, the timing and sequence of recruitment of individual pre- and post-synaptic components as synapses are formed and pruned in developing circuits.